Regulation of mammary gland development at puberty and the cyclical changes that occur in the adult mammary gland during pregnancy and lactation are highly dependent upon estrogen (E) and progesterone (P). During normal development the mammary gland undergoes reversible changes from hormone responsive to hormone refractory states. It is the long- term goal of this proposal to determine how these changes in responsiveness to E and P occur. Mammary stroma plays a critical role in mediating and inducing the proliferative effects of E and P in the mammary epithelium in vivo. The hypothesis being tested in this proposal is that the mitogenic effects of E and P are mediated by stroma-derived growth factors and extracellular matrix (ECM) molecules. We have developed a novel primary co-culture system of mammary epithelial cells and mammary stromal cells in serum-free medium in which the epithelial cells exhibit a proliferative response to both e and P. Using this culture system we will: 1) Identify the respective roles of mammary adipocytes and fibroblasts and characterize the nature of their interactions with epithelial cells; 2) Determine the role of growth factors, EGF, IGF-I, HGF, IGF binding proteins and their respective receptors (EGF-R, IGF-R and c-met) in E and P responsiveness in vitro and in vivo; 3) Define the role of stroma-derived ECM proteins (collagen IV, fibronectin and laminin) and their integrins in mediating E and P responsiveness; 4) Determine if estrogen responsiveness is determined by receptor (ER) isoform (alpha or beta) expression in epithelial and stromal cells. In order to understand E action in the stroma vs. epithelium, the temporal and cellular distribution of ER isoforms will be analyzed in relation to E responsiveness, both in vivo and in vitro. Determining the mechanisms that mediate epithelial-stromal cell interactions in normal mammary gland development and epithelial cell proliferation may provide a conceptual basis for novel approaches that focus on mammary stromal as a potential target for human breast cancer prevention and treatment.